Boothman, DA Abstract In non-small cell lung cancer (NSCLC), >80% tumors have elevated NQO1 levels 5- to 200-fold above associated normal tissues, while Catalase levels are expressed at extremely low levels in NSCLCs compared to all normal tissue in the body. We showed that unique and novel NQO1 bioactivatable drugs, -lapachone (ARQ761 in clinical trials) and a new drug, isobutyldeoxynyboquinone (IB-DNQ), are `bioactivated' by NQO1, resulting in massive H2O2-induced, apyrimidinic/apurinic (AP) site- and DNA single-strand break-mediated hyperactivation of poly(ADP- ribosyl) polymerase 1 (PARP1) that causes dramatic NAD+/ATP losses. -Lapachone is also an excellent radiosensitizer against cancer cells overexpressing NQO1. We hypothesize that exposure of NQO1+ NSCLC cancers with low dose ionizing radiation (IR), followed immediately with nontoxic NQO1 bioactivatable drugs (-lapachone or IB-DNQ for 2 h) will cause an elevated level of DNA lesions that hyperactivate PARP activity in cells that over-express NQO1. Low doses of IR or - lapachone, which are not able alone to cause a significant level of DNA lesions to hyperactivate PARP, will be used together. Since IR causes DSBs that are quickly repaired, this agent must be delivered before or at the same time as NQO1 bioactivatable drugs,18,19 which then must be applied for at least 2 h for maximal DNA lesion formation, PARP hyperactivation and metabolic alterations7,8,12 that inhibit DSB repair and cause synergistic lethality of NQO1+ NSCLC cells. Three aims will be performed: Aim 1: To define DNA repair inhibitory effects (on homologous recombination (HR) and non- homologous end joining (NHEJ)) after IR + -lapachone. (Yrs 0-5). Premise: Nonlethal, low doses of IR or -lap can be used to cause DSBs and PARP hyperactivation that depletes NAD+/ATP levels and inhibits DSB repair (NHEJ and HR) to enhance efficacy against NSCLC. Aim 2: To define the inhibition of carbon metabolism (glycolytic and TCA cycle inhibition due to NAD+/ATP losses) after IR + -lapachone (Yrs 0-5). Premise: PARP hyperactivation causes dramatic NAD+/ATP losses that greatly suppress glycolysis (via GAPDH inhibition) and TCA cycling by LDH inhibition and other NAD(P)H-dependent pathways to maximize efficacy against NQO1+ NSCLC. Aim 3: To elucidate the most efficacious use of IR + NQO1 bioactivatable drug combination against NQO1+ NSCLCs. (Yrs 0-5). Premise: IR + NQO1 bioactivatable drugs will induce DSBs + DNA lesions that hyperactivate PARP, lead to dramatic NAD+/ATP losses, inhibit DSB repair, greatly suppress glycolysis and TCA cycle metabolism that induces tumor-selective programmed necrosis. An outstanding research team will explore the effects of IR + NQO1 bioactivatable drugs on DSB repair, metabolism and efficacy against NSCLC xenograft models, leading to a clinical trial in 3-5 years.